RNA chain elongation on a chromatin template.

The rate of RNA chain elongation has been measured with DNA and chromatin as template. RNA propagation on chromatin is about 50% of the rate found with DNA. Kinetic experiments demonstrate that the inhibition is not due to interference with the addition of the nucleoside triphosphates. Analysis of the dependence of propagation on the Tm of DNA shows that the chromatin proteins interfere with the translocation of the RNA polymerase along the DNA template.     

Control of RNA synthesis by chromatin proteins.

The effect of chromatin proteins on template activity has been studied. Using both E. coli RNA polymerase and calf thymmus polymerase B we have measured the number of initiation sites on chromatin and various histone-DNA complexes. Chromatin can be reconstituted with histone proteins alone and this complex is still a restricted template for RNA synthesis. The removal of histone f1 causes a large increase in the template activity. Chromatin is then treated with Micrococcal nuclease and the DNA fragments protected from nuclease attack ("covered DNA") are isolated. Alternatively, the chromatin is titrated with poly-D-lysine, and by successive treatment with Pronase and nuclease, the DNA regions accessible to polylysine are isolated ("open DNA"). Both fractions were tested for template activity. It was found that RNA polymerase initiation sites are distributed equally in open and covered region DNA.     

Silence of the genes--mechanisms of long-term repression

A large fraction of genes in the mammalian genome is repressed in every cell throughout development. Here, we propose that this long-term silencing is carried out by distinct molecular mechanisms that operate in a global manner and, once established, can be maintained autonomously through DNA replication. Both individually and in combination these mechanisms bring about repression, mainly by lowering gene accessibility through closed chromatin structures.     

Muscle-specific activation of a methylated chimeric actin gene

To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.     

Regulation of mouse satellite DNA replication time.

The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.     

Design,Synthesis of Novel Lipids as Chemical Permeation Enhancers and Development of Nanoparticle System for Transdermal Drug Delivery

In the present study, we designed and developed novel lipids that include (Z)-1-(Octadec-9-en-1-yl)-pyrrolidine (Cy5T), 1, 1-Di-((Z)-octadec-9-en-1-yl)pyrrolidin-1-ium iodide (Cy5), (Z)-1-(Octadec-9-en-1-yl)-piperidine (Cy6T), and 1, 1-Di-((Z)-octadec-9-en-1-yl) piperidin-1-ium iodide (Cy6) to enhance the transdermal permeation of some selected drugs. Firstly, we evaluated the transdermal permeation efficacies of these lipids as chemical permeation enhancers in vehicle formulations for melatonin, ß-estradiol, caffeine, α-MSH, and spantide using franz diffusion cells. Among them Cy5 lipid was determined to be the most efficient by increasing the transdermal permeation of melatonin, ß-estradiol, caffeine, α-MSH, and spantide by 1.5 to 3.26-fold more at the epidermal layer and 1.3 to 2.5-fold more at the dermal layer, in comparison to either NMP or OA. Hence we developed a nanoparticle system (cy5 lipid ethanol drug nanoparticles) to evaluate any further improvement in the drug penetration. Cy5 lipid formed uniformly sized nanoparticles ranging from 150–200 nm depending on the type of drug. Further, Cy5 based nanoparticle system significantly (p<0.05) increased the permeation of all the drugs in comparison to the lipid solution and standard permeation enhancers. There were about 1.54 to 22-fold more of drug retained in the dermis for the Cy5 based nanoparticles compared to OA/NMP standard enhancers and 3.87 to 66.67-fold more than lipid solution. In addition, epifluorescent microscopic analysis in rhodamine-PE permeation studies confirmed the superior permeation enhancement of LEDs (detection of fluorescence up to skin depth of 340 μm) more than lipid solution, which revealed fluorescence up to skin depth of only 260 μm. In summary the present findings demonstrate that i) cationic lipid with 5 membered amine heterocyclic ring has higher permeating efficacy than the 6 membered amine hertocyclic ring. ii) The nanoparticle system prepared with Cy5 showed significant (p<0.05) increase in the permeation of the drugs than the control penetration enhancers, oleic acid and NMP.     

Direct detection of methylated cytosine in DNA by use of the restriction enzyme MspI.

The extent of methylation of the internal C in the sequence CCGG in DNA from various eukaryotic sources has been determined using the restriction enzyme MspI known to be specific for this sequence. The methylation of the CCGG sequence is reflected in the restriction pattern obtained by DNA treated with MspI and its isoschizomer HpaII and analyzed by gel electrophoresis. A direct method for detection 5-methylcytosine in the sequence CCGG has been deviced. DNA fragments obtained with MspI were radioactively labeled at their 5' ends and subsequently degraded to the corresponding 5'-deoxyribonucleoside monophosphates. 5 methylcytidylic acid has been found in most of the 5' ends of MspI fragments of calf thymus DNA (about 90%) indicating heavy methylation of the sequence CCGG in calf thymus DNA. The results also reveal a symmetric methylation of both strands at this sequence in calf thymus DNA. In contrast, the CCGG sequence in other eukaryotic DNAs from organisms like Neurospora, Drosophila and Herpes virus proved to be undermethylated at this sequence.     

A cost-effective maize ear phenotyping platform enables rapid categorization and quantification of kernels

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.     

A temperature-sensitive mutation in asparaginyl-tRNA synthetase causes cell-cycle arrest in early S phase

The Chinese hamster temperature-sensitive cell-cycle mutant ts24 was analyzed biochemically in order to determine the nature of this lesion. The inability of these cells to proceed through S phase at the restrictive temperature could be complemented by the addition of asparagine to the growth medium, and enzymological analysis showed that this line contains a temperature-sensitive asparaginyl-tRNA synthetase. Normal asparaginyl-tRNA synthetase activity was restored in cells transfected with cloned genomic DNA that overcomes the mutational defect. In corroboration with these results it was shown that a different temperature-sensitive asparaginyl-tRNA synthetase mutant isolated in another laboratory was blocked in S phase in a manner similar to that of ts24. While the mechanism by which asparaginyl-tRNA synthetase affects cell-cycle progression has not been elucidated, it can be shown that it is not mediated through alteration in overall levels of protein synthesis.     

Different data from different labs: lessons from studies of gene-environment interaction

It is sometimes supposed that standardizing tests of mouse behavior will ensure similar results in different laboratories. We evaluated this supposition by conducting behavioral tests with identical apparatus and test protocols in independent laboratories. Eight genetic groups of mice, including equal numbers of males and females, were either bred locally or shipped from the supplier and then tested on six behaviors simultaneously in three laboratories (Albany, NY; Edmonton, AB; Portland, OR). The behaviors included locomotor activity in a small box, the elevated plus maze, accelerating rotarod, visible platform water escape, cocaine activation of locomotor activity, and ethanol preference in a two-bottle test. A preliminary report of this study presented a conventional analysis of conventional measures that revealed strong effects of both genotype and laboratory as well as noteworthy interactions between genotype and laboratory. We now report a more detailed analysis of additional measures and view the data for each test in different ways. Whether mice were shipped from a supplier or bred locally had negligible effects for almost every measure in the six tests, and sex differences were also absent or very small for most behaviors, whereas genetic effects were almost always large. For locomotor activity, cocaine activation, and elevated plus maze, the analysis demonstrated the strong dependence of genetic differences in behavior on the laboratory giving the tests. For ethanol preference and water escape learning, on the other hand, the three labs obtained essentially the same results for key indicators of behavior. Thus, it is clear that the strong dependence of results on the specific laboratory is itself dependent on the task in question. Our results suggest that there may be advantages of test standardization, but laboratory environments probably can never be made sufficiently similar to guarantee identical results on a wide range of tests in a wide range of labs. Interpretations of our results by colleagues in neuroscience as well as the mass media are reviewed. Pessimistic views, prevalent in the media but relatively uncommon among neuroscientists, of mouse behavioral tests as being highly unreliable are contradicted by our data. Despite the presence of noteworthy interactions between genotype and lab environment, most of the larger differences between inbred strains were replicated across the three labs. Strain differences of moderate effects size, on the other hand, often differed markedly among labs, especially those involving three 129-derived strains. Implications for behavioral screening of targeted and induced mutations in mice are discussed.